Hybridization of molecules in biological samples using microarrays allows analysis and detection of multiple nucleic acid targets simultaneously. However, limitations of the method include its relatively low sensitivity. Often the DNA target sequence of interest (i.e. the sequence which must be detected) is only present in low abundance. Usually, nucleic acid targets of low copy e.g. 103, cannot be detected reliably by current methods.
Frequently, this necessitates the use of conventional polymerase chain reaction (PCR) techniques before the hybridization process, which restricts multiplicity. Several attempts have been made to amplify DNA targets by PCR directly on solid supports e.g., biochips, microbeads, and the like. However, these methods are either much less sensitive compared to that for conventional PCR, e.g. in tubes, or they can be used only for amplification of a very limited number of targets. Low sensitivity of PCR on solid supports may be merely due to the fact that, unlike the target, the primers are gathered in a volume that is much smaller than that of the reaction chamber so that only a small portion of the target can come into contact with the primers.
Some methods for performing PCR reactions on solid supports such as use on biochips or microbeads are not as sensitive as conventional PCR, or are restricted in the number of DNA sequences that can be detected. Moreover, often these methods require multiple steps and therefore are also slow, laborious, and expensive. Improved methods are needed.